Screening method and anti-tumor drug candidate obtained therefrom

ABSTRACT

A method for identifying a test compound that has an anti-tumoral effect whilst not significantly activating unrelated transduction signals, the method comprising the steps of: (a) selecting at least one test compound; (b) assaying the compound for anti-tumoral effect; (c) selecting at least one distinct intracellular event which is modulated, at least partially, by the GnRH receptor; (d) testing for the ability of said test compound not to modulate the selected intracellular event; and (e) selecting the test compound which selectively demonstrates an anti-tumoral effect and does not modulate said other selected intracellular event. A method according to any one of the preceding claims, the method comprising: (a) selecting at least one test compound; (b) determining whether the test compound activates signalling via Gαi; (d) testing for the ability of said test compound not to modulate signalling via Gαq; and (e) selecting the test compound which selectively activates signalling via Gαi and does not modulate signalling via Gαq. Compounds selected by the methods, including Ac-D-Nal(2)-D-4-ClPhe-D-Pal-Ser-1-MePal-D-IsopropylLys-Leu-IsopropylLys-Pro-D-AlaNH 2 , are useful in combating cancer and reproductive tissue hyperplasias.

This application is a national stage application under 35 U.S.C. § 371from PCT Application No. PCT/GB2003/003269 filed Jul. 22, 2003, whichclaims the priority benefit of Great Britain Application No. 0216963.9filed Jul. 22, 2002 and Great Britain Application No. 0310678.8 filedMay 9, 2003.

The invention relates to a new screening method for selecting an activecompound useful for the treatment of cancer or reproductive tissuehyperplasia.

GnRH (Gonadotropin-Releasing Hormone) is a decapeptide which plays apivotal role in the control of the reproductive axis of most knownspecies. It is released in a pulsatile manner from the hypothalamus andacts upon specific receptors in the anterior pituitary controlling therelease of luteinizing hormone (LH) and follicle-stimulating hormone(FSH). These two hormones are primary controllers of reproductivefunction.

A second form of GnRH, GnRH-II has been identified, in the mid-brain ofmany vertebrates. As a consequence the original form of GnRH has beenre-named GnRH I.

GnRH receptor expression has been demonstrated to be a vital componentin an autoregulatory cell proliferation process in a number of humanmalignant tumours, e.g. breast, ovary and endometrial cancers. Severalinvestigations have demonstrated some GnRH-receptor interacting ligands(agonists and antagonists) exert a dose-dependent anti-proliferativeeffect upon cell lines derived from reproductive tumours. Unlike GnRHreceptors in the anterior pituitary, the activation of these peripheralreceptors in reproductive tissues activates distinct signallingmodalities and seems to respond positively to both classical antagonistsand agonists. Despite the differences in cell signalling behaviour, thecloned sequences of the peripheral GnRH receptors appear identical tothat of the pituitary GnRH receptor. Therefore a method of selectingnovel GnRH receptor ligands which specifically activate thetumour-specific transduction pathways in cancerous tissue, as opposed tothose activated by GnRH in the pituitary, would be highly desirable.Such method would allow the selection of ligands of the GnRH receptorhaving enhanced selectivity and additionally reduce any side effectsinduced by GnRH treatment since we believe that current GnRH receptorbased therapeutics may be less than optimal by inducing tumourprogression from benign to more malignant steroid-independentphenotypes. Hence, traditional GnRH receptor based anti-tumour agentsact primarily by reducing serum levels of steroids, thus reducing thesteroid-dependent growth of the reproductive tumour. Protractedtreatment using the classical GnRH receptor-based therapeutics mayinitially induce regression of the tumour but eventually encourage thegrowth of more aggressive forms of the tumour.

GnRH agonists are extensively employed in the treatment of sex hormonedependent cancers. The predominant mode of action is believed to be viathe desensitization of pituitary gonadotropes. However, a substantialbody of evidence points to a direct inhibitory effect of GnRH analogueson these cancer cells. These effects appear to be mediated via the GαiG-protein in contrast to the predominant Gαq coupling in gonadotropes.Unlike Gαq coupling, Gαi coupling can be activated by both agonists andantagonists. This suggested to us that the receptor involved in thecancer cells may not be the gonadotrope Type I GnRH receptor. Theidentification of a Type II GnRH receptor in the marmoset which can beactivated by both GnRH agonists and certain antagonists suggested thatthis receptor may be the mediator of anti-proliferative effects incancer cells. However, extensive attempts to identify this receptor inman have been unable to demonstrate the existence of a conventional TypeII receptor transcript which would translate to a full-length functionalreceptor. The only full-length transcripts present in cancer cellsencode the Type I GnRH receptor. We have concluded, therefore, that thedifferent pharmacology (activity of agonists and certain antagonists) ofanti-proliferative effects are due to differences in the pharmacology ofactivation of the Type I receptors when coupled to differentintracellular signalling pathways (e.g. via Gαi). We report heredetailed studies on the molecular pathways mediating anti-proliferationand apoptosis in reproductive tract cancer cells, in benign hyperplasticcells and in HEK293 cells stably expressing the Type I GnRH receptor.These surprising results indicate for the first time that it is possibleto identify compounds which act via the Type I GnRH receptor and whichare anti-proliferative in tumour and hyperplastic cells but which at thesame time have substantially no effect on the Type I GnRHreceptor-mediated pathways of the type found in the pituitary, such asPLCβ activation. Without being bound by any theory, we believe thatthese compounds stabilise the active form of the GnRH receptor thatcouples to Gαi rather than Gαq; in other words, we believe that there isdifferential coupling Gαi and Gαq by the GnRH receptor and that thecompounds affect the Gαi-mediated pathway and not the Gαq-mediatedpathway.

It is therefore an object of the invention to provide a method foridentifying a test compound, preferably a GnRH receptor ligand, that hasan anti-tumoral effect whilst not significantly activating unrelatedtransduction signals, such as the Gαq-PLC-β cascade, which occur in thepituitary. In a first aspect, the method comprises the steps of:

-   a) selecting at least one test compound, which is preferably a Type    I GnRH receptor ligand;-   b) assaying the compound for anti-tumoral effect;-   c) selecting at least one distinct intracellular event which is    modulated, at least partially, by the GnRH receptor;-   d) testing for the ability of said test compound not to modulate the    selected intracellular event; and-   e) selecting the test compound which selectively demonstrate an    anti-tumoral effect and does not modulate at least said selected    intracellular event.

This method allows the distinction at an early stage, and in vitro,between test compounds which have potential as anti-tumoral compounds,but which activate unwanted transduction signals and, as such, should bedisregarded, and it allows the identification of test compounds whichare signal-specific and have a greater potential as a therapeutic agent.

Whilst the method can be used to distinguish between already known GnRHagonists/antagonists it can also be used to test other compounds. Inthis case the method of the invention can advantageously comprise apreliminary step wherein the test compound is tested for its capacity tobind to, and/or modulate the GnRH receptor. Advantageously, compoundswhich exhibit a high affinity for binding the GnRH receptor are used inthe assay method of the invention. Typically, the compound has a K_(D)for binding the GnRH receptor of between 0.1 and 10 nM, such as about 1nM. Whether or not a test compound binds to the GnRH receptor can bedetermined using methods well known in the art, such as a competitiveradioligand binding assay. The test compound may be any compound, buttypically it is an organic compound of between 100 and 10000 Daltons,preferably between 500 and 5000 Daltons. Typically, the test compound isa compound in a library of test compounds, such as those made usingcombinatorial chemistry techniques. Also, typically, the test compoundis a GnRH analogue.

It will be appreciated that in the methods described herein, which maybe drug screening methods, a term well known to those skilled in theart, the selected test compound may be a drug-like compound or leadcompound for the development of a drug-like compound.

The term “drug-like compound” is well known to those skilled in the art,and may include the meaning of a compound that has characteristics thatmay make it suitable for use in medicine, for example as the activeingredient in a medicament. Thus, for example, a drug-like compound maybe a molecule that may be synthesised by the techniques of organicchemistry, less preferably by techniques of molecular biology orbiochemistry, and is preferably a small molecule, which may be of lessthan 15000 daltons and which may be water-soluble. A drug-like compoundmay additionally exhibit features of bioavailability.

The term “lead compound” is similarly well known to those skilled in theart, and may include the meaning that the compound, whilst not itselfsuitable for use as a drug (for example because it is only weakly potentagainst its intended target, non-selective in its action, unstable,poorly soluble, difficult to synthesise or has poor bioavailability) mayprovide a starting-point for the design of other compounds that may havemore desirable characteristics.

The assay for anti-tumoral effects in step (b) of the method of thefirst aspect of the invention can be any one already used in the art forassessing the anti-tumoral activity of GnRH ligands. For example, asdescribed below, the ligands may be assessed for theiranti-proliferative effect on choriocarcinoma cell culture. It will beappreciated that the anti-tumoral activity of the compound may also bereadily assessed by determining whether the test compound is able toactivate a Type I GnRH receptor tumour-specific transduction pathwaywhich leads to an anti-proliferative effect. Typically, cells may betreated with the ligand, and its effect upon cell growth may be measuredas the ability to retard numerical cell growth over a period of, forexample, 5 days. Viable cells only will be counted, eg by counting thosecells which can efficiently exclude Trypan Blue dye.

In a preferred embodiment of the invention, the anti-tumoral effect isassayed by determining whether the test compound activates GnRHreceptor-mediated signalling via Gαi. Gαi is a heterotrimeric guaninenucleotide binding protein. Typically, it inhibits the enzyme adenylatecyclase. Also included by the term Gαi any member of the Gαi subfamily,including Gαo which is primarily found in the central nervous system.Whether or not a test compound activates GnRH receptor-mediatedsignalling via Gαi can be determined by methods well known in the art.For example, and as described in more detail in Example 1, this can bedone by determining whether the test compound is able to antagonizeforskolin (FSK) stimulated intracellular cAMP accumulation in a suitablecell (such as one which has sufficient Gαi and adenylate cyclase presentto facilitate experimentation) transfected with the Type I GnRHreceptor. Thus, in one embodiment, cells (eg human embryonic kidney(HEK) cells) stably expressing Type I GnRH receptor are pre-incubatedwith test compound then stimulated with forskolin, and cAMP is measuredcolorimetrically. This is compared with the amount of cAMP produced whenequivalent cells are treated with forskolin but not test compound. Thereis a reduction in cAMP levels in those cells where the test compound isone which is able to activate Gαi. Gαi activation may also be measuredby its direct association with the receptor, but this is less preferredcompared to methods that measure turnover (eg cAMP production). Theinvolvement of Gαi in a signal transduction event can be assessed usingpertussis toxin which inactivates Gαi; in other words, Gαi mediatedsignalling events are pertussis toxin sensitive.

JNK and/or p38α phosphorylation may also be used as a marker ofanti-tumoral effect, and Gαi signalling, and so can be used to assay theeffect of the test compounds (see, Example 1, and legend to FIGS. 7 and8). Similarly, a pertussin toxin (PTX)-sensitive ERK assay may also beused for example using similar methodology as described in relation toJNK and p38α phosphorylation (eg using antibodies selective forphosphorylated ERK1). Typically, cells are pre-incubated with 200 ng/mlPTX for 16 hours before stimulation. The stimulation period is generally4 to 5 mins. It is particularly preferred if test compounds are selectedwhich are able to produce a 2-fold or greater increase in Erk1/2phosphorylation above basal in a benign prostate hyperplastic cell.

In addition, it has been evidenced that GnRH ligands can induce apro-apopotic phenotype in tumour cells, and this may be another way ofassessing the anti-tumoral effect of a compound, albeit indirectly.Therefore the method may advantageously comprise the step of assayingthe GnRH ligand for the ability to induce a pro-apoptotic state incultured cells. In particular, determining whether or not a testcompound has a pro-apoptotic effect may be useful in connection withother means of assessing the anti-tumoral effect, since this assayprovides a strong indication if a test compound is drug-like in vivo. Inthe Example 1 the extracellular expression of phosphatidylserine (PS)phospholipid was the cellular event chosen as indicative of cellularapoptosis. Extracellular expression of PS can be readily determinedmaking use of labelled (eg fluorescently labelled) Annexin V whichselectively binds PS. Measurement of caspase or procaspase may be usefulin determining whether or not a test compound is proapoptotic. Suchmeasurments can be carried out in a high throughput screening formatwith appropriate enzyme substrates.

It will be appreciated that more than one method may be used to assesswhether the test compound has an anti-tumoral effect, and it may beadvantageous for more than one such method to be used. For example, thecapability of a test compound to activate Gαi may be measured using thecAMP accumulation assay described above, as well as the capability ofthe test compound to induce a pro-apoptotic state in cultured cells, forexample by measuring extracellular expression of PS by Annexin Vbinding.

Preferably, in step (c), the at least one distinct intracellular eventwhich is modulated, at least partially, by the GnRH receptor isactivation of the Type I GnRH receptor pathway which is activated inpituitary (the majority of pituitary function is Gαq mediated).Preferably, the distinct intracellular event is activation of the Gαqsignalling pathway. Gαq is a guanine nucleotide binding protein thatspecifcally activates the enzyme phospholipase Cβ. By Gαq we includeGα11 and Gα16, both of which can substitute for Gαq in various cellularackgrounds for the activation of phospholipase Cβ. In step (d) the testcompounds are tested for their ability not to modulate the selectedintracellular event (eg the Gαq signalling pathway) using methods wellknown in the art. For example, as described in Example 1, activation ofthe Gαq signalling pathway may be determined by measuring inositolphosphate production mediated by phospholipase Cβ (PLCβ) production(following Type I GnRH receptor-mediated activation of Gαq). PLCβ isinhibited by U73122 (available from Calbiochem Corporation, CA, USA).

At high levels of receptor stimulation, Gαi can cause PLCβ activation,therefore to show that PLCβ activation is via Gαq and not Gαi, PIX isused to inhibit Gαi, hence PTX-resistant inositol phosphate generationis indicative of Gαq-mediated activation of PLCβ activation. Thus,selecting compounds that do not activate Gαq may readily be done.

In step (e), those test compounds which selectively demonstrate ananti-tumoral effect and do not modulate at least said selectedintracellular event are selected for further study. It is particularlypreferred if compounds are selected which selectively activatesignalling via Gαi and does not modulate signalling via Gαq.

By “anti-tumoral effect” we mean an observable and/or measurable effect.When activation of GnRH receptor-mediated signalling via Gαi is used toassess the anti-tumoral effect, an observable and/or measurable effectmay be of downstream events as described herein. When induction of apro-apoptotic state in cultured cells is used to assess the anti-tumoraleffect, an observable and/or measurable effect may be of downstreamevents as described herein, including the measurement of PS on thesurface of dying cells, for example using annexin binding.

If JNK and/or p38α phosphorylation is used as a marker, an observableand measurable effect includes the use of fluorescent radioimmunoassayplate assays, or expression in cells of a marker gene, such asluciferase, whose expression is controlled at least in part by JNKand/or p38α-responsive reporter elements.

By “not modulating at least said selected intracellular event” we meanthat the test compound does not substantially modulate said event. Itwill be appreciated that a test compound may have an effect on the saidevent but that the effect is not substantial in the context of theinvention. For example, if the selected intracellular event isactivation of the Gαq signalling pathway, the test compound that isselected for further study is one which does not substantially activatethis pathway. It is particularly preferred if the test compound selectedis one which is an antagonist of a Type I receptor-mediated Gαqsignalling event. It is particularly preferred, however, that the testcompound is one with a high efficacy for activation of the Gαi pathway.

In a particular preferred embodiment of the invention, test compoundsare selected that have a high potency as an agonist of Gαi stimulation,with the compound having a low potency for Gαq stimulation. Thus, forexample, in relation to the given cell proliferation (mediated by Gαistimulation) or upon PTX-insensitive activation of PLC-β (mediated byGαq), the following table is helpful in understanding the selection of atest compound with the desirable properties.

Agonist potency - Gαi Agonist potency - Gαq Agent A HIGH Agent Y HIGHAgent B Agent X Agent C Agent Z Agent Z Agent A Agent Y Agent C Agent XLOW Agent B LOW

The most desirable agent would be Agent B since it has a high Gαipotency with the lowest potency at stimulating Gαq.

It will be appreciated that the test compounds selected are ones thatare not agonists of a Type I receptor-mediated Gαq signalling event, oronly have neligible capacity to activate Gαq pathways. For example,although GnRH I displays the capacity to attenuate cell growth, itpossesses the disadvantage of being able to cause gonadotropin secretionfrom the pituitary (ie is an agonist of a Type I receptor-mediated Gαqsignalling event). It will be appreciated, therefore, that the testcompounds selected behave like GnRH I at the peripheral tumour site butnot like GnRH I at the pituitary level.

Test compounds which are selected in the method typically are thosewhich exhibit a high ratio of Gαq EC₅₀/Gα_(i) EC₅₀. EC₅₀ means theconcentration of test compound which leads to 50% of the maximalresponse of Gαq or Gαi activation. The smaller the EC₅₀ the higher thepotency. It is preferred if test compounds with a Gαq EC₅₀/Gαi EC₅₀ratio of >10 are selected; more preferably those with a ratio a >50 areselected.

In a preferred aspect, the invention includes a method for selecting atest compound as a potential therapeutic agent or a drug-like compoundor a lead compound, the method comprising the steps of:

-   -   (a) determining whether the test compound activates GnRH        receptor-mediated signalling via Gαi;    -   (b) determining whether the test compound activates GnRH        receptor-mediated signalling via Gαq; and    -   (c) selecting the test compound which selectively activates        signalling via Gαi and does not activate signalling via Gαq.

It will be appreciated that by “does not activate signalling via Gαq” wemean does not significantly activate signalling via Gαq. Thus, the testcompound selected is one which has a negligible activity to activate Gαqpathways. Preferably, the method is used to select test compounds whichare suitable for treating cancer or hyperplasia of reproductive tissue,or are at least drug-like compounds or lead compounds for theseindications. The method or assay of the invention may also be used as aresearch tool to study and define the different activation states ofGnRH receptors and pinpoint the biological pathway triggered by aspecific state. This, in turn, has important and obvious medicalimplications for drug design, especially for selecting candidatecompounds which do not trigger undesirable side effects.

In a particularly preferred embodiment, assaying the compound foranti-tumoral effect (eg by determining whether the test compoundactivates GnRH receptor-mediated signalling via Gαi) and testing for theability of said test compound not to modulate the selected intracellularevent (eg determining whether the test compound activates GnRHreceptor-mediated signalling via Gαq or not) can be carried outsimultaneously on the same cell line using the above-mentioned methods.Typically, a number of cells would be divided between several growthplates and parallel Gαi (eg inhibition of forskolin-induced cAMPaccumulation) and Gαq (PLCβ activation) assays are performedsimultaneously. Advantageously, the method is one which can be used inhigh throughput screening systems, for example both of theaforementioned assays can be performed in commercially availableradioimmunoassay-based multiwell plate formats.

It will be appreciated that test compounds selected by the method mayfurther be tested for their anti-tumoral effect in model systems such asanimal model systems. Test compounds may still further be tested forefficacy in treating reproductive tumours or hyperplasias in clinicaltrials.

Additionally or alternatively, the test compounds may be used as thedrug-like compound or lead compound which is the basis for further drugdesign.

In a further embodiment the test compound is synthesised and may bepackaged and presented as a medicament and/or prepared into apharmaceutical composition.

The GnRH receptor ligands selected according to the method of theinvention are also an object of the invention.

It will be appreciated that preferred compounds selected using themethod of the invention are ones which (a) binds a GnRH receptor and (b)selectively activates signalling via Gαi and does not activatesignalling via Gαq. It is also preferred if the compounds selected areones which are GnRH receptor interacting agents which inhibitGnRH-mediated (via Gαq activation) gonadotropin release from thepituitary and directly affect tumour cell growth, in particular innon-steroid resistant tumours.

The ligand 135-25 having the formula:Ac-D-Nal(2)-D-4-ClPhe-D-Pal-Ser-1-MePal-D-IsopropylLys-Leu-IsopropylLys-Pro-D-AlaNH₂(wherein (Mepal is 1-Methyl-3-[3′-pyridyl]-alanine) has shown in vitropotent and selective anti-tumoral activity and has been selectedaccording to the method of the invention. Thus ligand 135-25 (alsocalled Ant 135-25 below), its use in a pharmaceutical composition andespecially in the manufacture of a drug for the treatment of tumours,sex-hormone dependent cancer or reproductive tissue hyperplasia isanother object of the invention.

Thus, a further aspect of the invention provides a compound selected bythe method of the invention for use as a medicament. Suitably, thecompound is packaged and presented for use as a medicament, for examplewith suitable instructions for its use in a patient.

A still further aspect of the invention provides a pharmaceuticalcomposition or formulation comprising a compound selected by the methodof the invention, and a pharmaceutically acceptable carrier. Thecarrier(s) must be “acceptable” in the sense of being compatible withthe compound of the invention and not deleterious to the recipientsthereof. Typically, the carriers will be water or saline which will besterile and pyrogen free.

Preferably, the formulation is a unit dosage containing a daily dose orunit, daily sub-dose or an appropriate fraction thereof, of the activeingredient.

A further object is a method of treatment of tumour, sex-hormonedependent cancer or reproductive tissue hyperplasia, such methodcomprising the step of administering an effective amount of ligand135-25 to a patient or other compounds selected according to the methodof the invention.

In addition, from the work described herein it is shown that compounds,such as 135-25, which bind a GnRH receptor and which selectivelyactivate signalling via Gαi and do not activate signalling via Gαq areuseful in treating these diseases. In particular, the compound is alsoone that has the ability to produce (at a concentration where it has itsmaximal effect) a 2-fold or greater increase in Erk1/2 phosphorylationover basal in a benign prostate hyperplastic cell such as a BPH-1 cellas described in FIG. 5, Example 1. Typically, the 2-fold or greaterincrease in Erk1/2 phosphorylation over basal is measured in cells whichhave been pretreated with PTX as described above (ie using a 16 hpre-incubation with 200 ng/ml PTX before application of the compound).

Thus, a further aspect of the invention provides a method of combatingtumours or reproductive tissue hyperplasias including specifcallyuterine fibroids in a patient, the method comprising administering acompound selected according to the invention or a compound which (a)binds a GnRH receptor; (b) selectively activates signalling via Gαi anddoes not activate signalling via Gαq; and (c) produces a 2-fold orgreater increase in Erk1/2 phosphorylation over basal levels in a benignprostate hyperplastic cell.

Preferably, the compound is able to produce a fold-increase in Erk1/2phosphorylation in benign hyperplastic cells very similar to thatproduced by GnRH I. Preferably, the compound is 135-25.

The method has uses in both veterinary or human medicine, therefore thepatient may be an animal (typically a mammal such as a dog, cat, horse,cow, sheep or pig) or a human. Preferably, the method is used to treathumans. Typically, the method is used to treat sex hormone-dependenttumours, such as tumours (or cancer) of the breast, prostate, ovary,endometrium or testicles.

It may be particularly advantageous to treat those patients who,following biopsy, are shown to have tumours with a high degree ofsteroid independency.

Existing therapies deprive tumours of the steroids required for theirgrowth while having perhaps a negligible potency with respect todirectly destroying the tumour cell. The compounds for use in themethods of the invention (and identifiable using the screening method)both have a capacity to control pituitary hormone secretion and directlydestroy the tumour cell. Neoplasms that grow independently of steroidsare resistant largely to existing therapies since their directanti-tumour efficacy has not been specifically selected for. Inaddition, conventional agonist based tumour therapeutics induce theunwanted side effect of “flare” of initial excess pituitary hormonerelease, which we believe will be avoided using the compounds describedand identifiable using the screening method herein. Because thecompounds act principally on the tumour, pituitary desensitisation doesnot occur and the compounds are less likely to lead to resistance to thecompound.

When hyperplasia is treated, it is particularly preferred to treatbenign prostate hyperplasia (BPH) or uterine fibroids (also known asleiomyomas).

The compounds for use in the methods of treatment of the invention willnormally be administered by any parenteral route, in the form of apharmaceutical formulation comprising the active ingredient, optionallyin the form of a non-toxic organic, or inorganic, acid, or base,addition salt, in a pharmaceutically acceptable dosage form. Dependingupon the disorder and patient to be treated, as well as the route ofadministration, the compositions may be administered at varying doses.In particular, the compounds for use in the methods of treatment of theinvention may be applied directly to the uterine lining.

In human therapy, the compounds for use in the methods of treatment ofthe invention can be administered alone but will generally beadministered in admixture with a suitable pharmaceutical excipient,diluent or carrier selected with regard to the intended route ofadministration and standard pharmaceutical practice.

The compounds for use in the methods of treatment of the invention canalso be administered parenterally, for example, intravenously,intra-arterially, intraperitoneally, intrathecally, intraventricularly,intrasternally, intracranially, intramuscularly or subcutaneously (whichis a preferred route of administration), or they may be administered byinfusion techniques. They are best used in the form of a sterile aqueoussolution which may contain other substances, for example, enough saltsor glucose to make the solution isotonic with blood. The aqueoussolutions should be suitably buffered (preferably to a pH of from 3 to9), if necessary. The preparation of suitable parenteral formulationsunder sterile conditions is readily accomplished by standardpharmaceutical techniques well-known to those skilled in the art.

Formulations suitable for parenteral administration include aqueous andnon-aqueous sterile injection solutions which may contain anti-oxidants,buffers, bacteriostats and solutes which render the formulation isotonicwith the blood of the intended recipient; and aqueous and non-aqueoussterile suspensions which may include suspending agents and thickeningagents. The formulations may be presented in unit-dose or multi-dosecontainers, for example sealed ampoules and vials, and may be stored ina freeze-dried (lyophilised) condition requiring only the addition ofthe sterile liquid carrier, for example water for injections,immediately prior to use. Extemporaneous injection solutions andsuspensions may be prepared from sterile powders, granules and tabletsof the kind previously described.

For parenteral administration to human patients, the daily dosage levelof the compounds of the invention will usually be from 1 to 1000 mg peradult (i.e. from about 0.015 to 15 mg/kg), administered in single ordivided doses. The physician in any event will determine the actualdosage which will be most suitable for any individual patient and itwill vary with the age, weight and response of the particular patient.The above dosages are exemplary of the average case. There can, ofcourse, be individual instances where higher or lower dosage ranges aremerited and such are within the scope of this invention.

The compounds for use in the methods of treatment of the invention canalso be administered intranasally or by inhalation and are convenientlydelivered in the form of a dry powder inhaler or an aerosol spraypresentation from a pressurised container, pump, spray or nebuliser withthe use of a suitable propellant, e.g. dichlorodifluoromethane,trichlorofluoromethane, dichlorotetrafluoro-ethane, a hydrofluoroalkanesuch as 1,1,1,2-tetrafluoroethane (HFA 134A3 or1,1,1,2,3,3,3-heptafluoropropane (HFA 227EA3), carbon dioxide or othersuitable gas. In the case of a pressurised aerosol, the dosage unit maybe determined by providing a valve to deliver a metered amount. Thepressurised container, pump, spray or nebuliser may contain a solutionor suspension of the active compound, e.g. using a mixture of ethanoland the propellant as the solvent, which may additionally contain alubricant, e.g. sorbitan trioleate. Capsules and cartridges (made, forexample, from gelatin) for use in an inhaler or insufflator may beformulated to contain a powder mix of a compound of the invention and asuitable powder base such as lactose or starch.

Aerosol or dry powder formulations are preferably arranged so that eachmetered dose or “puff” contains at least 1 mg of a compound of theinvention for delivery to the patient. It will be appreciated that heoverall daily dose with an aerosol will vary from patient to patient,and may be administered in a single dose or, more usually, in divideddoses throughout the day.

Alternatively, the compounds for use in the methods of treatment of theinvention can be administered in the form of a suppository or pessary,or they may be applied topically in the form of a lotion, solution,cream, ointment or dusting powder. The compounds of the invention mayalso be transdermally administered, for example, by the use of a skinpatch.

For application topically to the skin, the compounds for use in themethods of treatment of the invention can be formulated as a suitableointment containing the active compound suspended or dissolved in, forexample, a mixture with one or more of the following: mineral oil,liquid petrolatum, white petrolatum, propylene glycol, polyoxyethylenepolyoxypropylene compound, emulsifying wax and water. Alternatively,they can be formulated as a suitable lotion or cream, suspended ordissolved in, for example, a mixture of one or more of the following:mineral oil, sorbitan monostearate, a polyethylene glycol, liquidparaffin, polysorbate 60, cetyl esters wax, cetearyl alcohol,2-octyldodecanol, benzyl alcohol and water.

For veterinary use, a compound for use in the methods of treatment ofthe invention is administered as a suitably acceptable formulation inaccordance with normal veterinary practice and the veterinary surgeonwill determine the dosing regimen and route of administration which willbe most appropriate for a particular animal.

A further aspect of the invention provides use of a compound selectedaccording to the invention or a compound which (a) binds a GnRHreceptor; (b) selectively activates signalling via Gαi and does notactivate signalling via Gαq; and (c) produces a 2-fold or greaterincrease in Erk1/2 phosphorylation over basal levels in a benignhyperplastic cell in the manufacture of a medicament for combatingtumours or reproductive tissue hyperplasias.

It will be appreciated that the methods of treatment of the inventionmay comprise the administration to the patient of a further agent, suchas an anticancer agent.

Cancer chemotherapeutic agents include: alkylating agents includingnitrogen mustards such as mechlorethamine (HN₂), cyclophosphamide,ifosfamide, melphalan (L-sarcolysin) and chlorambucil; ethylenimines andmethylmelamines such as hexamethylmelamine, thiotepa; alkyl sulphonatessuch as busulfan; nitrosoureas such as carmustine (BCNU), lomustine(CCNU), semustine (methyl-CCNU) and streptozocin (streptozotocin); andtriazenes such as decarbazine (DTIC;dimethyltriazenoimidazolecarboxamide); Antimetabolites including folicacid analogues such as methotrexate (amethopterin); pyrimidine analoguessuch as fluorouracil (5-fluorouracil; 5-FU), floxuridine(fluorodeoxyuridine; FUdR) and cytarabine (cytosine arabinoside); andpurine analogues and related inhibitors such as mercaptopurine(6-mercaptopurine; 6-MP), thioguanine (6-thioguanine; TG) andpentostatin (2′-deoxycoformycin). Natural Products including vincaalkaloids such as vinblastine (VLB) and vincristine; epipodophyllotoxinssuch as etoposide and teniposide; antibiotics such as dactinomycin(actinomycin D), daunorubicin (daunomycin; rubidomycin), doxorubicin,bleomycin, plicamycin (mithramycin) and mitomycin (mitomycin C); enzymessuch as L-asparaginase; and biological response modifiers such asinterferon alphenomes. Miscellaneous agents including platinumco-ordination complexes such as cisplatin (cis-DDP) and carboplatin;anthracenedione such as mitoxantrone and anthracycline; substituted ureasuch as hydroxyurea; methyl hydrazine derivative such as procarbazine(N-methylhydrazine, MIH); and adrenocortical suppressant such asmitotane (o,p′-DDD) and aminoglutethimide; taxol andanalogues/derivatives; and hormone agonists/antagonists such asflutamide and tamoxifen. Other agents include paclitaxel, bombesin,gastrin releasing peptide (GRP) antagonists, and iressa (gefitinib; anEGF antagonist).

The invention will now be described in more detail by reference to thefollowing Figures and non-limiting Examples.

FIG. 1 shows the anti-proliferative effects of GnRHR-interacting ligandsupon human JEG-3 choriocarcinoma cells. Low cell density JEG-3 cellsubcultures were treated for 5 days continuously with the indicateddoses of GnRH I (a), GnRH II (b), antagonist 135-18 (c) or antagonist135-25 (d) added directly to Dulbecco's modified Eagles mediumsupplemented with 10% FCS, glutamine and penicillin/streptomycin.Ligands were replenished every 24 h. At the end of the stimulationperiod cells were removed from the growth plates with trypsin and mixedwith trypan blue dye (enters non-viable cells). Only viable cells weretherefore counted using a haemocytometer in quadruplicate for eachligand dose. The bars in each histogram represent the mean±s.e. mean ofn=4 experiments. Both GnRH I and GnRH II demonstrate a potent anddose-dependent anti-proliferative effect upon JEG-3 cells. Antagonist135-25 (Ant 135-25) displayed a similar potency and efficacy to GnRH Iand II whereas antagonist 135-18 (Ant 135-18) demonstrated a much lowerpotency and efficacy compared to 135-25, GnRH I and II.

FIG. 2 shows the anti-proliferative effects of GnRHR-interacting ligandsupon human benign prostate hyperplastic (BPH-1) cells. Low cell densityBPH-1 cell subcultures were treated for 5 days continuously with theindicated doses of GnRH I (a), GnRH II (b), Ant 135-18 (c) or Ant 135-25(d) added directly to RPMI 1640 medium supplemented with 10% FCS,glutamine and penicillin/streptomycin. Ligands were replenished every 24h. At the end of the stimulation period cells were removed form thegrowth plates by trypsin and mixed with trypan blue dye (entersnon-viable cells). Only viable cells were therefore counted using ahaemocytometer in quadruplicate for each ligand dose. The bars in eachhistogram represent the mean±s.e. mean of n=4 experiments. Both GnRH I,and to a lesser extent GnRH II, demonstrate a potent and dose-dependentanti-proliferative effect upon JEG-3 cells. Antagonist 135-25 displayeda similar potency and efficacy to GnRH I whereas Ant 135-18 demonstrateda much lower potency and efficacy compared to Ant 135-25 and GnRH I.

FIG. 3 shows the anti-proliferative effects of GnRHR-interacting ligandsupon human embryonic kidney (HEK293) cells expressing the Type I humanGnRHR. An identical methodology of measuring cell growth was employed asin FIG. 1. Due to the much greater level of GnRHR expression in theSCL60 cells (HEK293 cells stably overexpressing the human Type I GnRHR)a greater anti-proliferative efficacy is evident for GnRH I (a), II (b)and Ant 135-25 (d). However, Ant 135-18 (c) still displays a lowefficacy compared to the other ligands.

FIG. 4 shows the GnRH-induced apoptotic events in JEG-3 and BPH-1 cells.a) GnRH-induced plasma membrane translocation of phosphatidylserinemeasured using annexin V-FITC recombinant protein staining. All twelvepanels depict either phase contrast (1, 4, 7, 10), confocal lasermicroscope (2, 5, 8, 11) or phase contrast/confocal merged images (3, 6,9, 12). Panels 1 to 3 depict untreated control JEG-3 cells for cells inpanels 4 to 6 which have been treated with GnRH I (100 nM) for 24 h. Allcells have been exposed while live to an annexin V recombinant proteinconjugated to the FITC fluorophore. The annexin V will bind specificallyonly to phosphatidlyserine (PS) lipids usually only expressed on theintracellular side of the plasma membrane lipid bilayer. An early signof apoptosis is the translocation of these PS lipids from theintracellular side of the membrane to the outer aspect of the bilayer.As can be seen in panels 2 or 5 there is no significant expression ofannexin-V-reactive PS on the extracellular surface of the plasmamembrane. Panels 10 to 12 depict cells exposed to GnRH I for 48 h whilepanels 7 to 9 serve as unstimulated contemporaneous controls. It isevident in panel 11 that there is significant expression ofextracellular annexin-V-reactive PS on the JEG-3 cell membrane.Identical results were gained from similar GnRH I treatment of BPH-1cells (data shown in further figures). GnRH I induces an enhancedexpression of the pro-apoptotic proteases cleaved-caspase 3 andprocaspase 3 in both JEG-3 cells (b) and BPH-1 cells (c). The cellularlevels of caspase 3 and procaspase 3 were assessed by specificimmunoblotting of JEG-3 or BPH-1 whole cell (w-c) lysates from cellsstimulated continuously with GnRH I for the times depicted.

FIG. 5 shows that GnRHR-interacting ligands stimulate extracellularsignal regulated kinase (ERK1/2) in both BPH-1 and JEG-3 cells. a) GnRHI induces a strong and dose-dependent activation of ERK1/2 in BPH-1cells. The panel depicts a representative anti-phospho-ERK1/2 w-c lysateimmunoblot of stimulated BPH-1 cell extracts. The phospho-specificantisera identifies the activated form only of ERK1/2 while theanti-ERK2 sera reacts equally inactive and active forms of ERK2. Similarresults were achieved when similar studies were performed upon JEG-3cells, confirming other studies of GnRH-induced JEG-3 cell activation.Panel b) depicts the extent of ERK1/2 phosphorylation and henceactivation, in JEG-3 cells induced by several GnRHR-interacting ligands(all at 100 nM dose for 10 minutes). Each bar of the histogramrepresents the mean±s.e. mean of three to four experimental replicates.Both GnRH I and Ant 135-25 activate ERK1/2 to a similar extent, withGnRH II being less effective with Ant 135-18 the least effective. Panelc) depicts the extent of ERK1/2 activation in BPH-1 cells induced bystimulation with GnRH I, II, Ant 135-25 and Ant 135-18. A similarpattern to JEG-3 of ligand activation of the BPH-1 cells was observed.Each bar of the histogram represents the mean± s.e. mean of three tofour experimental replicates.

FIG. 6 shows the induction of inositol phosphate accumulation byGnRHR-interacting ligands in BPH-1 cells. BPH-1 cells were pre-incubatedwith ³H-myoinositol (1 μCi/ml) for 48 h prior to ligand stimulation (60minutes). Ligand stimulated inositol phosphate production was measuredin the presence of 10 mM LiCl. Panels a to d demonstrate that only highdoses of GnRH I and Ant 135-25 cause any significant accumulation ofinositol phosphates, suggesting that expression of the Type I GnRHR ispredominant in these cells. This has been confirmed by RT-PCR (data notshown), the poor activity of GnRH II upon inositol phosphateaccumulation is probably due to the lower sensitivity of the inositolphosphate assay as compared to the highly amplified ERK1/2 activationassay. The high doses though required of GnRH I to induce significantinositol phosphate accumulation suggested that the primary effector ofPLC-β activation, Gαq, may not be the mechanism by which the inositolphosphates accumulation is being induced. IP accumulation can be used asa read out/marker for Gαq activation since the activation of PLCβ isPTX-resistant. Panel e demonstrates that a Gαi mechanism may beresponsible for this high dose GnRH I effect. Hence the capacity of thehigh dose of GnRH I (50 μM) to induce an inositol phosphate accumulationwas abrogated by a 16 h pre-incubation of the BPH-1 cells with 200 ng/mlof pertussis toxin (PTX). In contrast the inositol phosphateaccumulation induced the potent cell mitogen lysophosohatidic acid (LPA)was insensitive to PTX pre-incubation suggesting that the LPA GPCR inthese cells can still stimulate PLC-β activation independently of Gαi.

FIG. 7 shows the GnRH-induced activation of JNK and p38mitogen-activated protein kinases in JEG-3 and BPH-1 cells. Theactivation of two other forms of mitogen-activated protein kinase (MAPK)was studied in JEG-3 and BPH-1 cells by overexpressing myc-taggedconstructs of JNK2 or p38α. Immunoprecipitation of these constructsafter GnRHR-ligand stimulation of either cell line allowed the degree ofkinase activation to be assessed using antisera specific for the active,phosphorylated, form of the kinase. In both cell lines there was apotent stimulation of these MAPKs, however there appeared to a be acell-dependency of the kinase stimulated, thus in JEG-3 cells only asignificant activation of the immunoprecipitated myc-JNK2 wasdemonstrated (Panel a, p38α data not shown) while in BPH-1 cells only asignificant activation of myc-p38α was detected (Panel b, JNK2 data notshown). In both panel a and b, the dose of GnRH I employed was 100 nM.The respective histograms in each panel represent the mean±s.e. mean ofthree experimental replicate time courses.

FIG. 8 shows the ligand-specific activation of JNK or p38 MAPK in JEG-3and BPH-1 cells. Panel a demonstrates that both GnRH I and Ant 135-25can efficiently activate the myc-JNK2 construct, measured by itsactivated phosphorylation status, while a similar cellular stimulationwith Ant 135-18 fails to efficiently activate JNK. The inset immunoblotsdepict the phosphorylation status of the immunoprecipitated JNK2increasing while the level of total unphosphorylated protein (detectedwith α-JNK antisera) remains unchanged. The histogram below depicts themean±s.e. mean of three experimental replicates of the above westernblot. Stimulation of BPH-1 cells with the same range of ligands (Panelb) yields a similar pattern of MAPK activation, but in this case p38α.Hence both GnRH I and Ant 135-25 effectively activate p38α while Ant135-18 does not. The inset immunoblots depict an increasingphosphorylation of p38α with no increase in total p38α protein. Thehistograin below depicts the mean±s.e. mean of three experimentalreplicates of the above western blot.

FIG. 9 shows that GnRH I and Ant 135-25 activate the Gαi-type G protein.In both BPH-1 and JEG-3 cells GnRH and Ant 135-25 can antagonizeforskolin (FSK) stimulated intracellular cAMP accumulation. Panels a andb respectively depict the effects of FSK, GnRH I and Ant 135-25 uponintracellular levels of cAMP in BPH-1 and JEG-3 cells measured using acalorimetric Biomol cAMP assay kit. As can be seen in panels a and b asingle stimulation with FSK (2 μM, 15 minutes) potently elevatesintracellular cAMP levels. However with increasing times of pre-exposure(10, 30, 60 minutes) to GnRH I or Ant 135-25 the extent ofFSK-stimulated cAMP accumulation was significantly reduced. The mostpotent inhibition of FSK-stimulated cAMP accumulation was seen with a 60minute pre-exposure for both GnRH I or Ant 135-25. In panels c (BPH-1)and d (JEG-3) the GnRH I and Ant 135-25 mediated inhibition ofFSK-stimulated cAMP accumulation (60 minute pre-exposure in each case)can be specifically inhibited with a 16 h pre-incubation of the cellswith 200 ng/ml of PTX. Each histogram in panels a to d depicts themean±s.e. mean of three to four experimental replicates of the cAMPaccumulation assay.

FIG. 10 shows that GnRH I and Ant 135-25 activation of JNK or p38 isdependent upon Gαi stimulation. Stimulation of JNK in JEG-3 cells andp38 in BPH-1 cells and its sensitivity to pre-exposure to PTX wasassessed by measuring the degree of activating phosphorylation ofimmunoprecipitated myc-JNK2 or myc-p38α. Panel a depicts arepresentative western blot of immunoprecipitated myc-JNK2 from JEG-3cells stimulated with either GnRH I (100 nM) or Ant 135-25 (100 nM). Thephosphorylation status of JNK was elevated by GnRH I and Ant135-25 withno significant alteration in total immunoprecipitated JNK levels(assessed by immunoblot with α-myc antisera). The increases in JNKphosphorylation however was abolished with the PTX pre-incubation (16 h,200 ng/ml). The histograms in panels b and c depict the mean±s.e. meanof three experimental replicates of the above western blot (panel a)experiments. Panel d depicts an identical experiment to that in panel aexcept that it is the phosphorylation status of immunoprecipitatedmyc-p38α that is being measured. Hence as with JEG-3 cells theactivation of the MAPK by both GnRH I and Ant 135-25 is acutelysensitive to the pre-exposure of PTX (16 h, 200 ng/ml). The histogramsin panels e and f depict the mean±s.e. mean of three experimentalreplicates of the above western blot (panel d) experiments.

FIG. 11 shows inhibition of GnRH I-mediated apoptosis by selectiveinhibition of JNK or p38 MAPKs. The contribution of the GnRH-mediatedJNK or p38 activation to the pro-apoptotic phenotype of the JEG-3 orBPH-1 cells (demonstrated in FIG. 4) was assessed by using a chemicalMAPK inhibitor. Panel a depicts phase confocal microscope images ofJEG-3 cells stimulated with 100 nM GnRH I in the presence or absence ofa high dose (20 μM of SB203580. This dose of SB203580 effectivelyabrogates the ability of GnRH I to stimulate JNK in these cells whilenot significantly affecting the activation of ERK1/2 MAPKs (data notshown). Unstimulated cells (1, 4, 7) demonstrate no annexin V FITCstaining indicative of a normal cell membrane morphology, with 48 h ofcontinuous GnRH I treatment cells begin to become anti-annexin V-FITCimmunoreactive (2, 5, 8) demonstrating that they have begun to enter anpro-apoptotic state. However when GnRH I is incubated in the presence ofSB203580 there is a significant reduction in extent of cells entering apro-apoptotic state (3, 6, 9). A similar experimental approach wasemployed for the transfer of this study to BPH-1 cells, however as theMAPK under study in these cells was p38α much lower dose of SB203580 (1μM) could be employed as the compound has a much greater inhibitorypotency against p38 compared to JNK. As with panel a no significanteffect of this SB203580 concentration was found upon GnRH-mediated ERKactivation in BPH-1 cells, yet this dose significantly blunted theGnRH-mediated activation of p38α (data not shown). As with the effect ofSB203580 co-incubation with GnRH in JEG-3 cells inclusion of thechemical inhibitor prevented GnRH from inducing a pro-apoptotic state inthe BPH-1 cells (compare 2, 5, 8 with 3, 6, 9).

FIG. 12 shows that Ant 135-25 possesses a distinct pharmacologicalprofile to Ant 135-18. Using two cell lines expressing a single GnRHreceptor population it can be demonstrated that Ant 135-25 does not seemto exert any significant biological activity at the Type II GnRHR,proposed to be the locus of GnRH action in peripheral tumour cells. Thehistograms in panels a and b represent inositol phosphate accumulationdata from HEK293 cells stably expressing the Type I GnRH receptor only(SCL60). In panel a GnRH I (10 nM) exerts a typical agonistic activityat its cognate receptor demonstrated by the significant increase inliberated inositol phosphates, however at the Type I GnRH receptor evenhigh doses of Ant 135-18 appears to have little classical agonisticcapacity. In panel b SCL60 cells are stimulated instead with Ant 135-25alongside GnRH I, yet again no classical agonistic activity isdemonstrated. In panels c and d, the liberated inositol phosphatesgenerated were measured in the proto-gonadotrope cells (αT4) stablyexpressing the marmoset Type II GnRH receptor (designated αT4-II). Inpanel c stimulation of the cells with GnRH II (10 nM) results in asimilar action to GnRH I upon the SCL60 cells, however Ant 135-18clearly exerts a dose-dependent agonistic activity upon these Type IIGnRH receptor expressing cells. In contrast, panel d, even high doses ofAnt 135-25 do not cause any liberation of free inositol phosphates. Inboth cell lines, SCL60 and αT4-II the free inositol phosphates measuredhere were liberated in a largely PTX-insensitive manner, suggesting thatto some extent Ant 135-25 may primarily exert only an agonistic effectin cellular environments where the GnRH receptor (Type I) ispredominantly coupled to Gαi-type G proteins.

EXAMPLE 1 Development of GnRH-Based Anti-Tumour Therapeutic Agents

Four GnRH receptor ligands (GnRH I, GnRH II, and two synthetic GnRHantagonists 135-18 and 135-25) have been screened.

Antagonist 135-18 has the formula:Ac-D-Nal(2)-D-4-ClPhe-D-Pal-Ser-Ile-D-IsopropylLys-Leu-IsopropylLys-Pro-D-AlaNH₂.

Antagonist 135-25 has the formula:Ac-D-Nal(2)-D-4-ClPhe-D-Pal-Ser-1-MePal-D-IsopropylLys-Leu-IsopropylLys-Pro-D-AlaNH₂.

Abbreviations used:

-   Ac-D-Nal(2) is acetyl-D-2-naphthylalanine;-   D-4-ClPhe is D-4-chlorophenylalanine;-   D-Pal is D-pyridylalanine;-   Ser is serine;-   MePal is methylpyridylalanine;-   Ile is isoleucine;-   D-IsopropylLys is D-isopropyllysine;-   Leu is leucine;-   Pro is proline;-   Ala is alanine.    Material and Methods

These are described in the legends to the Figures.

Results

GnRH and Ligand Analogues Mediates an Anti-Proliferative Effect UponCancerous Tissues

Continuous treatment of monolayers of either JEG-3 humanchoriocarcinoma, human benign prostatic hyperplasia (BPH-1) or HEK293cells stably expressing the rat type I GnRH receptor (SCL60) resulted ina retardation of the cellular reproductive growth of all three of thesecell types. Each cell type was plated at an initial minimal confluency(10-20%) to allow for 5 days of normal continual cell growth that wouldnot result in 100% cell confluency by day 5. Each ligand was incubated,in triplicate for every experimental concentration, with the cellmonolayers for five days with replacement of the ligand every 12 h. Atthe end of the stimulation period the number of viable cells wasestimated by their capacity to prevent uptake of Trypan Blue stain. InFIG. 1 it can observed that there are clear dose-response relationshipsfor the inhibition of cellular growth of the JEG-3 cells by GnRH I(panel a), GnRH II (panel b) and the antagonist 135-25 (Ant135-25: paneld). However the antagonist 135-18 (Ant135-18), chemically similar toAnt135-25, failed to demonstrate an anti-proliferative effect of asimilar magnitude to that generated by GnRH I, GnRH II or Ant135-25.Thus these data suggest that unlike recent reports (Grundker et al(2002) J. Clin. Endocrinol. Metab. 87, 1427-1430) it appearspharmacologically that GnRH I and GnRH II exert a similar effect uponcell proliferation. In addition it appears unlikely that theanti-proliferative GnRH-based effect occurs via a Type II GnRH receptor(GnRHR) stimulation as Ant135-18 has been demonstrated to possess a highdegree of partial agonistic activity upon Type II GnRHRs cloned fromseveral species (Ott et al (2002) Mol. Endocrinol. 16, 1079-1088) andyet it fails to exert an anti-proliferative action greater than that byAnt135-25 which fails to show any agonistic activity at the Type IImarmoset GnRHR.

GnRH and Ligand Analogues Mediates an Anti-Proliferative Effect UponHyperplastic Tissues

Employing an identical growth and stimulation methodology to thatdescribed in the previous paragraph and in FIG. 1 we exposed thehyperplastic cell line, BPH-1, to the same panel of GnRH ligands andantagonistic analogues (FIG. 2). Reminiscent of FIG. 1 both endogenousGnRH agonists exert a similar mode of action with respect to theirdose-dependent inhibition of BPH-1 cell growth over the five daystimulation period. However in slight contrast to the JEG-3 cells GnRH Iappears to be more potent that GnRH II in its capacity to arrest cellgrowth. This result directly contrasts recent assertions that indeed aType II GnRH receptor or a specific GnRH II ligand effect plays asignificant role in the anti-proliferative effect of GnRH ligands upontumour cells. As with the ligand effects upon JEG-3 cells Ant135-25exerts an anti-proliferative effect upon BPH-1 cells with a similarpotency to that of GnRH I while Ant135-18 once again demonstrates a lowant-proliferative potency thus reinforcing the concept that theanti-proliferative effect occurring in these cells is not through adirect action upon a functional human Type II GnRH receptor.

GnRH and Ligand Analogues Mediates an Anti-Proliferative Effect UponNon-Cancerous Non-Hyperplastic Tissue

We further investigated the nature of the ligand-receptor specificity ofthe anti-proliferative effects of GnRH receptor systems by studying ourligand panel effects upon a model cell background, i.e. SCL60 HEK293cells stably expressing the rat Type I GnRHR. Upon continuous treatmentof the SCL60 cells with GnRH I (FIG. 3, panel a) there was a dramaticreduction in the cells growth rate and significant loss of total cellnumber. As with the BPH-1 cells the GnRH II ligand appeared less potentthan GnRH I at arresting the SCL60 cell growth. The two classical GnRHRantagonist behaved in a similar manner as in the previous two cell linesin that Ant135-18 proved to be relatively ineffective at inhibiting theSCL60 cell proliferation while Ant135-25 had nearly an as efficaciousaction as either GnRH I or GnRH II. Compared to either JEG-3 or BPH-1cells there was considerably greater inhibition of cell growth and agreater degree of detectable cell death apparent from 48 h onwards. Weattribute this greater effect of the GnRHR ligands upon the SCL60 cellsto the much greater level of receptor expression in the SCL60 cells.Only minimal levels of cell surface receptor expression was noted inJEG-3 cells and BPH-1 cells (not in excess of 200 specific cpm forI¹²⁵-His⁵-Tyr⁶-GnRH I) while up to a 10-fold greater expression level isdemonstrated in SCL60 cells.

GnRH Induces the Generation of Pro-Apoptotic States in Cancerous andHyperplastic Cells

Thus we have shown that upon continuous GnRH ligand stimulation ofeither JEG-3 or BPH-1 there is a retardation of cell growth, however wewere interested as to whether there was any genuine induction ofcellular apoptosis recently reported to occur in several tumour celllines (Soon et al (2002) J. Clin. Endocrinol. Metab. 87, 4580-4586). Tothis end we investigated whether GnRH ligand stimulation of either cellline resulted in the generation of classic signals of apoptosis.Initially we measured the effects of GnRH stimulation upon theultrastructural integrity of the cells plasma membrane. A welldocumented early event in apoptosis is a reversal in the polarity ofmany plasma membrane molecules such as phosphatidylserine (PS). At anearly stage of cellular apoptosis there is a significant translocationof PS from the inner to the outer envelope of the plasma membrane.Employing the high affinity of annexin-V protein for exposed PS wetested as to whether protracted GnRH exposure of JEG-3 cells resulted inthe expression of annexin-V-reactive PS on the outer envelope of cellsstimulated with GnRH I. Unstimulated JEG-3 cells after 24 h ofsub-culture failed to demonstrate any extracellular membrane PS as uponincubation for 1 h with annexin-V pre-conjugated to the FITC fluorophore(1:100 dilution) there was no significant green fluorescence associatedwith the cells (FIG. 4, panel a, images 1-3). Stimulation of cells for24 h and then incubation of them with the annexin-V-FITC demonstratedthat additionally there was no significant expression of extracellularmembrane PS (FIG. 4, panel a, images 4-6). However with a greater periodof continuous stimulation (48 h) there was evident a considerable amountof external membrane annexin-V-reactive PS (FIG. 154, panel a, images10-12). Contemporaneously cultures unstimulated cells in contrast failedto exhibit any external membrane annexin-V-FITC staining (FIG. 4, panela, images 7-9). Thus after only 48 h of continuous GnRH I stimulation(100 nM) there was induction of plasma membrane reversal as indicated bythe presence of extracellular envelope PS. Similar results were obtainedfrom BPH-1 cells for a similar period of GnRH I stimulation (data notshown). In addition to the generation of early plasma membrane PSreversal we also were able to demonstrate additional pro-apoptoticevents induced in the cells by protracted GnRH I exposure. Hence westudied the generation of pro-apoptotic caspase enzymes involved in celldegradation in many tissues. Crude whole-cell lysate extracts were madefrom JEG-3 or BPH-1 cells and the cellular levels of either pro-caspase3 or its cleaved and active bi-product, caspase-3 were measure byspecific immunoblots. In JEG-3 cells stimulated with 100 nM GnRH Icontinuously there was a significant elevation in the cellular levels ofpro-caspase 3 evident from 24-48 h after initial ligand stimulation(FIG. 4, panel b). The generation and elevation of cellular levels ofactive caspase-3 took longer to emerge and were only significantlyevident between 48-72 h of GnRH I incubation. A similar pattern to thatin JEG-3 cells of the time-dependent increases in pro-caspase andcleaved caspase-3 in BPH-1 cells during GnRH I stimulation was seen inthe BPH-1 cells (FIG. 4, panel c), yet there appeared to be a more rapidonset on the generation of cleaved caspase-3 and a greater basal levelof pro-caspase-3 at the initiation of GnRH continuous stimulation.

GnRH Receptor Activation Activates Stress-Activated Protein KinasePathways in Hyperplastic and Cancerous Tissue

It has been demonstrated by many research groups (Kang et al (2000) Mol.Cell. Endocrinol. 170, 143-151; Kimura et al (1999) Cancer Res. 59,5133-5142) that upon stimulation of tumour cell lines there is a potentand protracted stimulation of the extracellular signal-regulated kinase(ERK) isoforms of the mitogen-activated protein kinase (MAPK) family.Coincident with this activation of ERK however is the demonstration ofthe anti-proliferative action of the GnRH analogues despite the welldocumented generally proliferative effects of ERK activation in manytissues (Gutkind (1998) J. Biol. Chem. 273, 1839-1842). Thus a paradoxexists in that the GnRH analogues appear to arrest cell growth andproliferation but potently activate ERK isoforms. Recent data has alsodemonstrated that an inhibitory effect upon epidermal growth factorreceptor (EGFR) activity (Grundker et al, (2001) Endocrinology 142,2369-2380) is in part responsible for the anti-proliferative action ofGnRH analogues. However there is significant evidence for the generationof a long lasting ERK activation in peripheral GnRH-responsive tumourtissues and indeed in our experimental paradigms we did indeed observe areproducible GnRH I-induced activation of ERK1/2 kinases in both JEG-3and BPH-1 cells (FIG. 5). Compared to the stimulation of otherendogenous G protein coupled receptors, e.g. the LPA-responsive EDG-typereceptors the degree of ERK1/2 activation was relatively small (data notshown). As with the effects upon cell proliferation we noted thatcompared to GnRH I, GnRH II and Ant135-25 the potency of Ant135-18 tostimulate ERK1/2 activation was considerably less (FIG. 5, panels b andc). In addition to the activation of ERK1/2 MAP kinases in peripheraltumor cells many reports have demonstrated a lack of inositol phosphateturnover induced by GnRH stimulation. Indeed we also noticed that withlow doses of GnRH and its analogues (up 1 to 1 μM) there was noappreciable inositol phosphate turnover (FIG. 6, panels a to d). Howeverat high doses (up to 50 μM) GnRH I, GnRH II and Ant 135-25 all displayeda small capacity to induce inositol phosphate accumulation. The dosesthat activated this turnover would suggest that rather than PLC-βactivation by its cognate G protein (Gαq) that activation of inositolturnover was being mediated by Gβγ subunits of another G protein, e.g.Gαi. Therefore in FIG. 6 panel e, we demonstrated that the GnRH-inducedminimal inositol phosphate turnover was sensitive to pre-treatment withpertussis toxin (PTX) while the more robust inositol phosphate turnoverinduced by LPA treatment (also activating a Gαq-coupled receptor) wascompletely insensitive to the pertussis toxin. Thus it appears that incorroboration with preceding reports there is negligible inositolphosphate turnover induced in peripheral tumor cells by GnRH. However atmuch higher doses there seems to be a PTX-sensitive capacity tostimulate inositol phosphate turnover presumably by the Gβγ-mediatedactivation of PLC-β.

Continuous treatment however of either JEG-3 or BPH-1 cells with 1 mMLPA failed to significantly attenuate the cell growth and invariablycaused a slight elevation in cell number after five days of continuoustreatment. Thus it appears that the ability of the GnRH-inducedstimulation of either JEG-3 or BPH-1 cells is not correlated to theiranti-proliferative action. We additionally assessed whether the GnRHstimulation of either JEG-3 or BPH-1 resulted in the significantactivation of any other of the MAPK isoforms. Using specific antiseraagainst the kinase active form of ERK5 (or BMK) we observed no specificactivation of this form of MAPK however using antisera specificallyrecognising the active forms of either c-Jun N-terminal kinase (JNK) orp38 MAPK we noted that in JEG-3 cells there was a potent, yet delayedGnRH-induced activation of JNK and a similarly slow inset activation ofp38 in the BPH-1 cells. The level of activation of the endogenousstress-activated protein kinases (SAPKs) was relatively small butgreater than the GnRH-activated ERK levels in these cells. To furtherinvestigate the validity of the observed SAPK activation we transfectedthe tumour cell lines with myc-tagged JNK2 or p38α MAPK isoforms,stimulated the cells with GnRH and then immunoprecipitated with anti-mycsera and western blotted the immunoprecipitates for total JNK/p38protein and active JNK/p38. In FIG. 7 (panel a) GnRH causes ademonstrable, time-dependent and protracted activation of theimmunoprecipitated JNK2. Activation of JNK2 typically only occurredafter 30 minutes of GnRH I stimulation. In the BPH-1 cells thestimulation with GnRH I also resulted in a protracted activation of theimmunoprecipitated myc-p38α. As with the activation of the JNK in theJEG-3 cells there was a considerable delay in the onset of the p38activation unlike the ERK signalling events that typically occur andpeak within 20 minutes of GnRH ligand application (FIG. 7, panel b).

GnRH-Induced Activation of Stress-Activated Protein Kinase Pathways isInvolved in the Induction of a Pro-Apoptotic State

We investigated whether there was a connection between the capacity ofGnRH to activate the SAPK pathways and the observed generation of theearly signs of apoptosis, e.g. the PS transfer from the internal face ofthe plasma membrane envelope to the external face, thus making itreactive with the annexin-V-FITC protein conjugate. To this end weemployed the SAPK inhibitor SB203580 which at low doses (1 μM) acts as apotent inhibitor of p38 SAPK activity yet at higher doses (20 μM) exertsan additional inhibitory activity upon the JNK family of SAPK proteins(Mangoura et al (2000) J. Dev. Neurosci. 18, 693-704). Co-incubation ofJEG-3 cells with 20 μM SB203580 and GnRH I for 48 h resulted in asignificant reduction in the degree of annexin-V-FITC staining of theexternal face of the plasma membrane (FIG. 11, panel a, 4-6 compared to7-9). There was observed to be no significant difference in the generalgrowth patterns and gross morphology of the cells treated with SB203580compared to those treated with GnRH alone or those unstimulated. DMSOvehicle controls were performed for the SB203580 treatment yet thesedemonstrated no significant effect upon cell growth or morphology (datanot shown). In parallel experiments, but using a lower more specificdose of SB203580 (1 μM) BPH-1 cells were co-incubated with GnRH I withor without SB203580 for 48 h. As demonstrated in FIG. 11, panel b,images 4-6, there was a significant induction of annexin-V-FITCreactivity on the outer plasma membrane envelope that was almostcompletely abrogated by the co-treatment of GnRH I plus 1 μM SB203580.As with the experiments upon JEG-3 cells there was no significantobservable change in cell morphology or growth rates either with theSB203580-treated cells or the DMSO vehicle-treated cells. Thus itappears that the inhibition of the GnRH-induced SAPK pathways in theJEG-3 cells and BPH-1 cells can attenuate the capacity of GnRHRactivation to inhibit cellular proliferation in these two cell lines.

GnRH Ligand Activation of the SAPK Pathways in JEG-3 and BPH-1 CellsOccurs Via a Gαi-Based Receptor Mechanism

We have demonstrated that in both the tumour cell line, JEG-3 and thehyperplastic cell line, BPH-1, that upon Type I GnRHR activation thereis a profound activation of SAPK pathways and that these protein kinasesare linked to the generation of the early signs of programmed cell deathin both models. It has been shown by many experimental groups that the Gprotein coupling GnRH receptors expressed in peripheral tumour tissuesis aberrant compared to that in the pituitary setting. In the pituitarythe primary G protein coupling event of the stimulated GnRHR is via theGαq-type G proteins leading to the increase in intracellular Ca²⁺ andthe eventual activation of protein kinase C isoforms. However incontrast in peripheral tissues the primary GnRHR G protein couplingevent appears to be via the pertussis toxin-sensitive Gαi G proteinpathway. This occurs despite the fact that in these cell lines there isdemonstrably only one form of the GnRH receptor, i.e. the Type I GnRHR.Thus we tested whether in our experimental paradigms that GnRH ligandactivation of the cells resulted in the stimulation of the SAPK pathwayspreviously implicated in the generation of the early signs of apoptosisthrough a Gαi type G protein pathway. In addition we were interested inelucidating whether there was any correlation between the GnRH I-likeanti-proliferative capacity of some GnRH-based peptide antagonists(Ant135-25) and their ability stimulate such atypical Gαi G proteinpathways.

We first demonstrated that there was actual activation of Gαi protein inboth cell lines upon GnRH and Ant 135-25 stimulation. Thus we noted thatwhen cyclic adenosine monophosphate levels (cAMP) were measured using acommercially available fluorescent assay system (Biomol) the ability offorskolin (1 μM in JEG-3 and 3 μM in BPH-1, sufficient to give a 50%R_(max) response in each case) was blunted with extended cellularpre-treatment times (10 to 60 minutes) with either 100 nM GnRH I or Ant135-25 (FIG. 9, panel a-BPH, panel b-JEG). In FIG. 9 panels c (BPH) andd (JEG) the ability of the 60 minute GnRH I or Ant 135-25 pre-treatmentsto inhibit the forskolin-mediated cAMP accumulation was attenuated by a16 h pre-treatment with 200 ng/ml PTX. Therefore it appears that bothligands can efficiently activate the adenylate cyclase inhibitoryactivity of Gαi in both cell models tested.

The ability of either GnRH I or Ant135-25 to stimulate the SAPK pathwaysin JEG-3 or BPH-1 cells was estimated and whether this SAPK pathwayactivation was via an atypical Gαi-type G protein pathway. Henceemploying the transfected myc-JNK2 in JEG-3 cells or myc-p38α in BPH-1cells the degree of JNK or p38 stimulation by Ant135-25 was assessed. Asdemonstrated in FIG. 10 (panels a and d) representative western blotsshow that upon both GnRH I (100 nM, 30 minutes) or Ant135-25 (100 nM, 30minutes) stimulation there is a similar level of JNK2 or p38α activationin JEG-3 and BPH-1 cells respectively. With a pre-incubation of 16 hwith 200 ng/ml of pertussis toxin (PTX) there was observed to be asignificant inhibition of either GnRH I or Ant 135-25 to activate theSAPK pathways suggesting that indeed both ligands are activatingGαi-type G protein pathways. Panels b and c in FIG. 10 demonstrate themean data compiled in JEG-3 cells for the inhibitory action of PTXpre-treatment upon GnRH I- or Ant135-25-induced JNK2 activation. Panelse and f show similar data gathered from p38α activation experimentsperformed in BPH-1 cells again showing that the GnRH I and Ant135-25stimulation of p38α occurs through a Gαi PTX-sensitive pathway.

Antagonist 135-25 and not Antagonist 135-18 Potently Stimulates theActivation of SAPK Pathways in JEG-3 and BPH-1 Cells

We have demonstrated that upon continuous stimulation with either GnRH Ior Ant135-25 that both JEG-3 and BPH-1 cells slow down in their growthrates and demonstrate signs of an early apoptotic state. However whenthese experiments were performed in parallel using an agent chemicallyrelated to Ant135-25, i.e. Ant 135-18, the anti-proliferative effectseen with this agent was minimal despite its similar structure to themore potent Ant135-25. Therefore we investigated whether this phenomenonof the low anti-proliferative potency of Ant135-18 resided in itscapacity to activate the Gαi-SAPK pathways. When tested for its capacityto activate either JNK2 in JEG-3 cells or p38α in BPH-1 cells Ant135-18demonstrated a dramatically lower efficacy than GnRH I or Ant 135-25with respect to activating the SAPK isoforms (FIG. 8). Therefore itappears that Ant135-25 like GnRH I can adequately activate the Gαi-typepathway in JEG-3 or BPH-1 cells while the chemically related Ant135-18has a much lower potency with respect to this form of atypical GnRHreceptor activation. Therefore we would suggest that this inability ofAnt135-18 to induce a productive coupling between the GnRHR and the GαiG protein pathway in these model cells lies at the centre of its pooranti-proliferative potency.

Antagonist 135-25 Demonstrates a Selective GnRH Receptor ActivationProfile

We have demonstrated that despite a high degree of similarity betweenAnt135-18 and Ant135-25 there is a significant difference in theircapacity to stimulate certain forms of GnRH receptor activity, hence thegeneration of an anti-proliferative effect upon three both tumorous andhyperplastic cell lines. Recent reports have suggested that the atypicalGnRH receptor pharmacology observed in peripheral reproductive tumorlines is due to the expression of a Type II human GnRH receptor similarto that originally cloned by Millar et al (2001) Proc. Natl. Acad. Sci.USA 98, 9636-9641. We have previously shown that upon mammalian Type II(marmoset) and non-mammalian GnRH receptors that classical GnRH Type Ireceptor antagonists possess varying degrees of partial agonisticactivity. However if indeed the peripheral anti-proliferative actions ofGnRH and related ligands upon the model cells used in this present studythen one would expect that Ant135-18 would possess a much greateranti-proliferative efficacy than Ant135-25 as the former displays a muchgreater partial agonist activity upon non-mammalian (data not shown) andmammalian Type II GnRH receptors (FIG. 12). Thus when compared in HEK293cells expression the rat Type I GnRH receptor (SCL60) both antagonistsAnt135-18 and Ant135-25 exert a classical antagonist activity anddemonstrate no partial agonistic activity (FIG. 12, panels a and b).However when the two antagonists are compared in a cellular backgroundexpressing solely the marmoset Type II, i.e. αT4 gonadotropes stablyexpressing the marmoset Type II GnRH receptor (Millar et al (2001) Proc.Natl. Acad. Sci. USA 98, 9636-9641), Ant 135-18 demonstratedconsiderable partial agonistic activity (FIG. 12 panel c) whileAnt135-25 showed no partial activity at all upon the Type II marmosetGnRH receptor (FIG. 12, panel d). Therefore it is extremely unlikelyfrom a pharmacological and molecular biological viewpoint that theanti-proliferative actions of GnRH I or Ant135-25 are occurring viastimulation of a novel human Type II GnRH receptor.

The ability of the four major agents under study at activatingectopically expressed Type I or Type II GnRHR were tested (FIG. 12). Theclassical signal transduction cascade activated by GnRHR stimulation isthe Gαq-PLC-β cascade. Both Type I and II receptor can stimulate theGαq-PLC type-G-protein and activate PLC-β which cleavesphosphatidylinositol bisphosphate (PIP₂) into inositol trisphosphate(IP₃), other lower inositol phosphates (IP_(n)) and diacylglycerol(DAG). The major function of IP₃ is to elevate intracellular Ca²⁺ whileDAG causes the activation of specific PKC (protein kinase C) isoforms.Measuring the generation of these IP_(n)s, we demonstrated that bothType I and Type II GnRHRs were activated by the endogenous ligands (TypeI and II GnRH). However, only the Type II receptor was activated by135-18 with no activation of the PLC-β signal at the Type I receptorevident (data not shown). In contrast the chemically-similar agent135-25 exhibited no IP_(n)-generating capacity at either ectopicallyexpressed Type I or II GnRHRs (FIG. 12).

Panels a and b of FIG. 12 depict the generation of free cytoplasmic³H-myo-inositols on the plasma membranes of HEK293 cells expressing theType I GnRHR and of αT4 gonadotropes expressing the marmoset Type IIGnRHR. Prior to agonist stimulation the respective cells were incubatedin inositol-free growth media supplemented with 1 μCi/ml ³H-myoinositolfor 48 h. G protein coupled receptor (GPCR)-induced activation of PLC-βresults in the cleavage of PIP₂ (incorporating tritium ³H) into freelower inositol phosphates (IP_(n)s). Stimulation of the Type I or TypeII GnRHRs with GnRH I or GnRH II respectively yields an increase in freetritiated inositol phosphates. However stimulating either receptor withantagonist 135-25 (10 μM-1 μM) failed to generate significant freeinositol phosphate. Therefore it appears that the 135-25 antagonist isspecifically unable to activate Gαq-type G-proteins despite displaying ahigh binding affinity for both Type I and II GnRHRs (data not shown) anda high anti-proliferative efficacy.

Therefore it appears that 135-25 possesses the potential to attenuatetumour cell proliferation without having any significant effect uponother GnRHR signalling modalities. As the two antagonists 135-25 and135-18 employed differ only slightly in their chemical composition (i.e.a MePal residue in position 5 for 135-25 of a Ile for 135-18), yet havedifferent efficiencies against tumour cell proliferation theirphysio-chemical differences may indicate a valuable target formodification at this site to enhance the compounds anti-tumour capacityof GnRH ligands.

Discussion

In this current study we have demonstrated that like endogenous GnRHligands such as GnRH I, typically described ‘antagonists’ can act asagonists with respect to peripheral tumor cells due to the alteredpharmacological profile of the receptor in these cells. Thedemonstration of the elevated potency of Ant 135-25 over Ant 135-18 withrespect to the antiproliferative effect appeared to due to the relativeabilities of the two peptides to stabilize an active form of thereceptor able to couple productively to Gαi. Thus the designation of Ant135-25 as a GnRH receptor ‘antagonist’ is somewhat spurious as this onlydescribes its ability to activate/stabilize the Gαq-preferringform/state of the Type I GnRH receptor. Hence we have described inessence that Ant 135-25 exerts a potent antiproliferative action due toits selective ability to activate a Gαi-coupling form of the Type I GnRHreceptor while being unproductive upon Gαq-coupling forms of thereceptor.

Initial hypotheses concerning the nature of the divergence in signallingbetween GnRH-responsive sites in the pituitary and those in peripheralreproductive tissues suggested that the receptor present in theseperipheral sites was of a distinct nature those in the gonadotrope.However recent evidence has suggested that the two receptor sites areindeed the same, yet their associated signal transduction mechanisms areclearly distinct (Chegini et al (1996) J. Clin. Endocrinol. Metab. 81,3215-3221; Yin et al (1998) Life Sci. 62, 2015-2023; Chatzaki et al(1996) Cancer Res. 56, 2055-2065). This dichotomy also extended to theeffects of agonist and antagonists of the pituitary form of GnRHsignalling as proliferation of both endometrial and ovarian cancer cellscan be inhibited by agonistic and antagonistic analogues of GnRH (Emonset al (1997) Trends Endocrinol. Metab. 8, 335-362). A solution to thisproblem was proposed by Imai et al ((1996) J. Clin. Endocrinol. Metab.77, 132-137) in which they speculated that the G protein ai thatpossibly couples the GnRH receptor to the effector may be responsiblefor the differences in response between peripheral tumors and theanterior pituitary. Interestingly it has been shown that, as with ourstudy, functional LPA-mediated Gαq-coupling activity is present in thesetumor cells (Grundker et al (2001) Endocrinology 142, 2369-2380), thus apathophysiological loss of G protein cannot explain the paradoxicalchange in receptor signalling. In the present study little GnRH-mediatedactivation of Gαq was observed but other groups have recentlydemonstrated that in other reproductive tumor lines such as Ishikawacells, GnRH can induce coupling to Gαq (Grundker et al (2001)Endocrinology 142, 2369-2380). However this group found that this extantGαq signalling was not involved in the generation of theantiproliferative effect of GnRH. Thus it is probable that thefunctional signalling complexes containing the GnRH receptors inperipheral tumor sites are able to coerce the receptor into specific Gαicoupling and those complexes in the pituitary are not. Whatever theprotein intermediates responsible for this shift in functionality it isclear that additional receptor-interacting factors can dramaticallyalter the way in which a given ligand can direct its signal to theintracellular environment and with clearly distinct physical endpoints.This differential coupling of the receptor therefore necessitates us tore-evaluate our terminology with respect to the nature of the ligandsinteraction with the GnRH receptor at these peripheral tumor sites. Thuswe have shown that while Ant 135-25 can be adequately described as anantagonist at the anterior pituitary level with respect to theactivation of the Gαq-based signalling mechanisms it behaves as a trueagonist in the peripheral cells as it appears almost equally effectiveat stimulating the existing Gαi-coupled Type I GnRH receptors. Inaddition we have clearly demonstrated that a separation between thesetwo effects at the periphery and the pituitary can be engineered byalteration of the primary sequence of the GnRH receptor peptide ligand.Therefore conversion of the single amino acid difference between Ant135-18 and Ant135-25 resulted in a dramatic elevation of the agent'spotency at the Gαi-coupled peripheral GnRH receptor while not changingits ability to functionally inhibit the action of GnRH at the pituitaryGαq-coupled receptor.

The use of GnRH analogues, both agonist and antagonists, is now widelyaccepted with applications in gynaecology, reproductive medicine andoncology. The mechanisms of action of the majority of these therapeuticsis based on an inhibition of the anterior pituitary and the gonads.Classical ‘antagonistic’ GnRH receptor ligands have an advantage overGnRH agonistic peptides due to the fact that they inhibit the secretionof gonadotropins and sex steroids immediately after first applicationachieving more rapid therapeutic effects than GnRH agonists that requirerepeated administration (Schally, A. V. (1999) Peptides 20, 1247-1262).The repeated exposure to agonistic agents is required as they need toinduce a functional downregulation of the anterior pituitary GnRHsignalling system. For conditions such as prostate cancer GnRH classical‘antagonist’ molecules are preferable to agonists specifically as theyavoid the so-called ‘flare’ of the disorder which occurs inapproximately 10-20% of patients when agonists are given as singleagents (Schally, A. V. & Comaru-Schally, A. M. (1997) Hypothalamic andother peptide hormones. In: Holland J. F., Frei, E., Bast, R. C., Kufe,D. E., Morton, D. L., Weichselbaum, R. R., eds. Cancer Medicine, 4^(th)Ed. Baltimore: Williams & Wilkins p1067-1086.). Pre-existingantagonistic therapies for reproductive tumors involve theadministration of Cetrorelix which has been demonstrated in somecircumstances to attenuate the growth of androgen-dependent prostatecancers (Redding et al (1992) Cancer Res. 52, 2538-2544; Korkut et al(1991) Proc. Natl. Acad. Sci., USA 88, 884-888). However considerablyhigher doses of Cetrorelix were required to attenuate the growth ofandrogen-resistant tumor cells (Jungwirth et al (1987) Prostate 32,164-172; Jungwirth et al (1997) Eur. J. Cancer 33, 1141-1148) suggestingthat its direct antiproliferative efficacy could be significantly lowerthan its ability to attenuate anterior pituitary gonadoptropin releasewhich would deprive the androgen-sensitive tumor of itsgrowth-sustaining steroid. Thus it appears that the capacity to directlyattenuate tumor growth may be specifically desirable to agents aimed atrelieving aggressive androgen-insensitive prostate tumors, hence ourattempts at elevating the direct antiproliferative potency of theantagonistic agent Ant 135-25. Indeed in our hands Cetrorelix wassignificantly less potent than Ant 135-25 when compared in theirabilities at stimulating PTX-sensitive/Gαi-dependent MAPK isoformactivation (data not shown). In addition it appears that Cetrorelix maynot possess a true and profound antiproliferative effect upon allreproductive tumors expressing GnRH receptors, e.g. theantiproliferative effect of triptorelin (GnRH agonist) upon LNCaPprostate cells was efficiently inhibited in the presence of Cetrorelix(Ravenna et al (2000) J. Androl. 21, 549-557). Thus it is possible thatCetrorelix has a significantly lower antiproliferative potency thanTriptorelin and therefore in its presence acts as a functionalantagonist of its action. In other experimental paradigms otherclassical GnRH antagonists, e.g. Antide, have been shown to functionallyinhibit the antiproliferative effects of classical GnRH receptoragonists (Kang et al (2000) Endocrinology 141, 72-80). Thus it ispossible that the majority of existing therapeutics do not have asignificantly potent direct anti-tumor effect, which would be desirablefor steroid-resistant neoplasms. Their poor potency may be due to theirlow potency with respect to stabilizing/inducing the Gαi-preferringconformation/state of the Type I GnRH receptor. We have therefore shownthat by modifying an antagonist with a low direct antiproliferativepotency (Ant 135-18) by one residue we have significantly elevated itsantiproliferative potency by allowing it to potently activate theGαi-type of GnRH signalling seen in peripheral reproductive tumors. Anagent such as Ant135-15 is believed to display several properties makingit superior to current GnRH-based peptide treatment of reproductiveneoplasms, i.e. not being an agonist there is no initial disease‘flare-up’ (Schally, A. V. & Comaru-Schally, A. M. (1997) Hypothalamicand other peptide hormones. In: Holland J. F., Frei, E., Bast, R. C.,Kufe, D. E., Morton, D. L., Weichselbaum, R. R., eds. Cancer Medicine,4^(th) Ed. Baltimore: Williams & Wilkins p1067-1086.), its inhibitoryaction at the pituitary will decrease serum levels of sex steroidsthereby attenuating steroid-sensitive neoplasm growth and finally itsenhanced direct anti-tumor effect would be directly cytotoxic tosteroid-resistant cells potentially present in the neoplasm.

EXAMPLE 2 Treatment of Breast Cancer

A patient presenting with breast cancer is administered from 1 to 100 mgof the compound Ant 135-25 intravenously daily for a week.

EXAMPLE 3 Treatment of Benign Prostastatic Hyperplasia (BPH)

A patient presenting with BPH is administered from 1 to 100 mg of thecompound Ant 135-25 intravenously daily for a week.

EXAMPLE 4 Treatment of Prostate Cancer

A patient presenting with prostate cancer is administered a depot forthe delivery of the compound Ant 135-25.

1. A method of combating tumors or reproductive tissue hyperplasia in apatient, the method comprising administering the compoundAc-D-Nal(2)-D-4-ClPhe-D-Pal-Ser-1MePal-D-IsopropylLys-Leu-lsopropylLys-Pro-D-AlaNH₂.
 2. A methodaccording to claim) 1 wherein the tumor is a sex-hormone dependentcancer.
 3. A method according to claim 1 wherein the tumor is a prostatetumor.
 4. A method according to claim 1 wherein the reproductive tissuehyperplasia is benign prostate hyperplasia or uterine fibroids.